Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITION FOR THE INHIBITION OF
VASCULAR OCCLUSION
Background of the Invention
The present invention is generally in the area of methods for
inhibiting or preventing vascular smooth muscle cell proliferation, or
restenosis, following vascular intervention or injury, such as angioplasty,
vascular bypass surgery, organ transplantation, or other vascular
intervention or manipulation.
Angioplasty, surgery and other vascular intervention are
complicated by an accelerated arteriopathy characterized by rapid growth
of cells into the lumen within a short period of time which is severe
enough to jeopardize the blood flow to distal organs.
Vascular bypass surgery has been widely used to correct stenotic
and occluded blood vessels, as when plaques develop on the surface of
blood vessels in atherosclerosis. In bypass surgery, one or more healthy
blood vessels are grafted into the occluded vessels at either end of the
occlusion to shunt blood around the stenotic or occluded vessel to re-
establish a sufficient blood supply to the tissue whose blood supply is
endangered by the stenosis or occlusion. This surgery often successfully
revascularizes the endangered tissue.
In recent years, angioplasty has been developed as an alternative
treatment to bypass surgery, especially in patients who have been
diagnosed early in the development of stenosis or occlusion of blood
vessels due to the abnormal laying down of plaque on the endothelial wall
of a blood vessel. Angioplasty typically involves guiding a catheter which
is usually fitted with a balloon or expandable metal mesh up through an
artery to the region of stenosis or occlusion and the brief inflation, one or
more times, of the balloon or wire mesh to push the obstructing
intravascular material or plaque up against the endothelial wall of the
vessel, thereby compressing and/or breaking apart the plaque and
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reestablishing blood flow. However, angioplasty treatment can injure the
vessel, especially when the balloon is overinflated or the mesh
overextended, causing a variety of undesirable results, such as denudation
(removal) of the endothelial cell layer in the region of the angioplasty,
dissection of part of the inner vessel wall from the remainder of the vessel
with accompanying occlusion of the vessel, or rupture of the tunica intima
layer of the vessel.
Though the hallmark of the rapidly accelerating lesions following
angioplasty, vascular bypass grafting, and organ transplantation is the
proliferation of smooth muscle cells and their accumulation within the
tunica intinza, it is the loss of normal endothelial function that heralds
these events and may stimulate them to occur. The arterial endothelium
serves as a transport barrier, a biochemical filter and as a regulator of
many vascular phenomena. The most potent vasodilators,
thromboresistant compounds and inhibitors of smooth muscle cell
proliferation, are endothelial derived. Vascular smooth muscle cell
accumulation within the intima ceases with restoration of the endothelium
(Schwartz et al., Am. J. Pathol., 81: 15-42 (1975); Fishman et al., Lab.
Invest., 32: 339-51 (1975)) and regression of intimal hyperplasia is
maximized where endothelial restoration is maximized (Bjornsson et al.,
Proc. Natl. Acad. Sci. USA, 88: 8651-8655 (1991)). Confluent, and not
exponentially growing, endothelial cells produce a series of compounds
that are the most potent vasodilators, inhibitors of spasm, and inhibitors
of smooth muscle cell proliferation. Heparan sulfate proteoglycan
produced by the endothelial cells has multitudinous effects on the smooth
muscle cells including interfering with binding of heparin-binding growth
factors (Nugent et al., Circulation Research, 73: 1051-1060 (1993), which
are known to stimulate vascular smooth muscle cell growth (Nugent et
al., Circulation Research, 73: 1051-1060 (1993); Castellot et al., J. Cell
Biol., 90: 372-9 (1981)). It appears, therefore, that restoring the
endothelial monolayer of a blood vessel restores the agents or compounds
responsible for biochemical control of vascular cell proliferation.
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Other efforts at limiting the undesirable proliferative and disease
states of vascular endothelium have focused on the isolated administration
of analogs of endothelial compounds. Certain drugs, such as heparin, are
especially effective inhibitors of vascular smooth muscle cell proliferation
in tissue culture and animal models of arterial diseases precisely because
they mimic the activity of natural endothelial-derived compounds like
heparan sulfate proteoglycan, Edelman, E.R. & Karnovskv, M.J. Circ.
89: 770-776 (1994). However, despite cell culture and small animal data
supporting the regulatory role of heparin-like compounds, exogenous
heparin preparations have shown no benefit in human trials. For
example, when patients were randomized to heparin or dextrose infusion
over the first 18 to 24 hours post angioplasty, 41.2% of the heparinized
patients and only 36.7% of the dextrose infusion patients had evidence for
restenosis (Ellis et al., Am. Heart. J., 117: 777-782 (1989)). Moreover,
bleeding complications were twice as frequent in the heparinized group.
In another trial, angioplasty patients injected subcutaneously with heparin
at 10,000 IU/day had 2.5 fold more restenosis and significantly more
ischemic complications than patients treated in the standard fashion
(Lehmann et al., J. Am. Coll. Cardiol., 17: 181A (abstract) (1991)).
Non-heparin endothelial compounds such as nitric oxide and the
prost,aglandins are potent regulators of a range of biologic effects
involving smooth muscle cells. Inhibitors of these compounds have been
shown to control intimal hyperplasia following experimental vascular
injury (Cooke et al., Curr. Opin. Cardiol., 7: 799-804 (1992); Moncada
et al., N. Engl. J. Med., 329: 2002-2012 (1993); McNamara, et al.,
Biochem. Biophys. Res. Comm., 193: 291-296 (1993)). This is indicative
that the vascular endothelium is a powerful regulator of the blood vessel
= wall, not because of the production and secretion of one compound alone,
but because of its presence as an intact unit. While concerns have been
raised as to the difference in protocols allowing for benefit in animals and
worsening of disease in man (Edelman et al., Circulation, 89: 770-776
(1994)), it, appears likely that a single endothelial product, such as
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heparin, is incapable of replacing the entire endothelium in the control of
complex human vascular lesions.
Accordingly, there is a need for means and methods of promoting
healing of vascular tissue and controlling vascular muscle cell
proliferation (hyperplasia) to prevent restenosis of blood vessels after
angioplasty, vascular bypass, organ transplantation, or vascular disease
without the risk of rapid reocclusion.
It is therefore an object of the present invention to provide a
means of preventing occlusion following vascular intervention.
It is a further object of the present invention to provide methods
for understanding and controlling the mechanisms of restenosis and
vascular occlusion.
Summary of the Invention
Compositions and methods for inhibiting excessive intimal growth
(hyperplasia) and/or vascular smooth muscle cell proliferation
(hyperplasia) following intravascular intervention or injury are disclosed.
In the preferred embodiment, endothelial cells are present in or on a
biocompatible, biodegradable or non-biodegradable matrix, which allows
the cells to reside and secrete products into the surrounding tissue or into
the matrix, from which they can diffuse into the surrounding tissue. The
endothelial cell-matrix composition is juxtaposed with the target blood
vessel, which might be injured, for example, from angioplasty, surgery,
transplantation or atherosclerosis, to inhibit subsequent restenosis or
occlusion of the blood vessel.
More specifically, one aspect of the invention involves the use of a
composition for inhibiting mechanisms involving restenosis of a blood vessel
following injury to vascular tissue in a region of the blood vessel of a
patient in
need of treatment thereof. The composition includes a biocompatible matrix
having seeded therein or thereon dissociated endothelial cells implantable at
a site
exterior to and adjacent to or at the injury to or blockage of vascular
tissue. The
endothelial cells are provided in an amount effective to inhibit smooth muscle
cell
proliferation at the site of the injury or blockage.
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The matrix may be formed of a material selected from the group
consisting of polyhydroxy acids, polyorthoesters, polyanhydrides, proteins,
carbohydrates or polysaccharides, polyphosphazenes, polyalkylene oxides and
combinations thereof.
Alternatively, the matrix may be formed of a material selected from the
group consisting of ethylene vinyl acetate, polyvinyl alcohol, silicone,
polyurethane, non-biodegradable polyesters, polyethyleneoxide-
polypropyleneoxide and combinations thereof.
The matrix may further comprise biologically active compounds selected
from the group consisting of anti-inflammatory agents, prostaglandin,
prostanoids, compounds regulating the renin-angiotensin axis, tyrosine kinase
inhibitors, immunosuppressants, vitamins, glucocorticoids, anti-oxidants, free
radical scavengers, peptide hormones, angiogenic and angiogenic inhibitory
factors, and combinations thereof.
Brief Description of the Drawings
Figure 1 is a graph of the growth (measured as cell number) of bovine
aortic endothelial (BAE) cells (open circles) and Chinese Hamster Ovary 745
cells (controls which do not produce regulators of smooth muscle cell
proliferation) (dark circles) on GelfoamTM (The Upjohn Co., Kalamazoo, MI)
collagen matrix over time (days).
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Figure 2 is a graph of inhibition (% control) of binding (dark
circles) and mitogenesis (dark squares) of the potent vascular cell growth
promoter 1bFGF for vascular smooth muscle cells by endothelial cell-
Gelfoam* conditioned media, compared with inhibition of bFGF binding
(open circles) and mitogenesis (hatched squares) for vascular smooth
muscle cellls by CHO-745 cell-Gelfoam conditioned media.
Figure 3 is a graph of the ratio of the area of the tunica intima to
the area of the tunica media after balloon denudation of the endothelium
(BI) when the balloon injured arteries were exposed to Gelfoam alone
(Gel), Gel.foam engrafted with CHO-745 cells (CHO), Gelfoam
containing heparin (Hep), and Gelfoam containing endothelial cells (EC).
Figure 4 is a graph showing cell proliferation within both the
tunicae intima and media, when exposed to either Gelfoam engrafted
with endothelial cells (EC), Gelfoam with heparin (Hep), Gelfoam
engrafted with CHO-745 cells (CHO), Gelfoam control or untreated
following balloon angioplasty (BI), comparing the % proliferating cells
for the tunica intima (open bar) and tunica media (dark bar).
Detailed Description of the Invention
The compositions described herein for inhibition of intimal
hyperplasia consist of a matrix, most preferably a hydrogel, seeded with
endothelial cells which can be xenografts, allografts or autografts, which
is implanted at a site near to or at the region of hyperplasia at the time of
vascular intervention or upon diagnosis of restenosis or other occlusion of
a blood vessel.
Compositions to Inhibit Intimal Hyperplasia
The compositions described herein are used to inhibit undesired
= response to vascular injury that includes hyperplasia of smooth muscle
vascular cells which occurs in response to injury to the endothelial tissue
of blood vessels, for example, as a result of angioplasty, coronary artery
bypass used to open a stenotic or occluded vessel. Vascular smooth
muscle cell hyperplasia triggered by the injured endothelium can result in
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restenosis of the blood vessel due to the excessive proliferation of
vascular smooth muscle cells.
Cells
Endothelial cells are isolated by standard methods, for example, as
described by Gimbrone, M. Culture of vascular endothelium. Progress y
Hemostasis and Thrombosis 3:1-28 (1976). Cells can be obtained at the
time of the procedure using standard biopsy techniques, whether the
procedure is angioplasty, open field surgery or for diagnostic purposes.
The cells can be dissociated with collagenase or trypsin and seeded
directly into a matrix as described below for immediate implantation, or
cultured in vitro as required to generate the number of cells to be
implanted. The cells are typically seeded into or onto the matrix at a
density of between approximately 103 and 1011 cells/cm3. Cell densities
can be determined using visual methods or a Coulter counter. The
effective amount of cells and matrix to be administered is that which
prevents or inhibits hyperplasia at the site of vascular injury.
Other cells which are genetically engineered, using
oligonucleotides resulting in ribozyme-mediated cleavage of targeted genes
or regulatory elements, or antisense to block transcription of targeted
genes, or sequences encoding defective, missing or insufficient genes, can
also be used. For example, cells transfected with genes for synthesis of
heparan sulfate, regulators of heparan sulfate, nitric oxide synthase,
growth factors, cytokines and other vasoregulatory products could be
seeded into the matrix and implanted. Genes can be transfected into the
cells using standard methodology such as viral vectors, microinjection and
calcium phosphate precipitation.
Other materials can also be administered via the matrix, including
anti-inflammatory agents, prostaglandins, prostanoids, angiotensin and -
related compounds, tyrosine kinase inhibitors, immunosuppressants,
vitamins, glucocorticoids, anti-oxidants, free radical scavengers, peptide
hormones, angiogenic and angiogenic inhibitory factors.
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The Matrix
The endothelial cells are seeded onto or into a biocompatible
matrix which is suitable for implanting in contact adjacent to or at the site
of the vascular injury, for example, by wrapping around the blood vessel.
The matric can be in the form of a gel, foam, suspension, microcapsules,
solid polylneric support, or fibrous structure. The matrix may also serve
in a physically supporting role. There is no specific requirement as to =
thickness, size or shape. When the cells are seeded within the matrix, it
is preferred that the matrix be sufficiently porous to allow free, diffusion
of nutrients and gases into the matrix to maintain cell viability, while
allowing the secretory products of the cells to diffuse out of the matrix
into the surrounding tissue in roughly physiologically quantities. The
matrix may also serve to protect non-autologous cells from immune
attack.
Preferably, the matrix is a biodegradable material, such as a
synthetic polymer degrading by hydrolysis, for example, polyhydroxy
acids like polylactic acid, polyglycolic acid and copolymers thereof,
polyorthoesters, polyanhydrides, proteins such as gelatin and collagen, or
carbohydrates or polysaccharides such as cellulose and derivatized
cellulosesõ chitosan, alginate, or combinations thereof, so that over the
course of several days or weeks after implant.ation of the matrix material,
the matrix: gradually disappears. In a preferred embodiment, the matrix is
a hydrogel, defined as a matrix wherein typically approximately 90% by
weight of the matrix is absorbed water. Hydrogels can be formed by
ionic or covalent crosslinking of a variety of water soluble polymers such
as polyphosphazenes, polysaccharides such as alginate, and proteins such
as gelatin. As demonstrated in the following examples, a presently
= preferred matrix material is purified gelatin-based GelfoamTM (The
Upjohn Co., Kalamazoo, MI) surgical sponge.
The use of biodegradable matrices eliminates the need for surgery
to remove undegraded implanted matrix, after regression of the
hyperplasia and healing of the endothelial injury. However, synthetic
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non-biodegradable matrices may also be used. Useful materials include
ethylene vinyl acetate, polyvinyl alcohol, silicone, polyurethane, non-
biodegradable polyesters, and polyethyleneoxide-polypropyleneoxide, and
tetrafluoroethylene meshes (Teflon ).
Attachment of the cells to the matrix can be enhanced by coating
or chemically modifying, attaching or derivatizing the polymer with a
material such as collagen, laminin, fibronectin, fibrin, basement
membrane components, and attachment peptides (RGD and many others
are known and described in the literature).
Culturing of Cells in Matrix
The matrix material seeded with endothelial cells can be cultured
in vitro to promote cell growth throughout the matrix. At various times
during incubation of the seeded matrix in the culture medium, samples of
seeded matrix can be removed and examined, for example, by cell
staining and microscopic observation, to determine the extent of cell
growth and proliferation in the matrix, as well as production of total
sulfated glycosaminoglycans and, specifically, heparan sulfate.
When the cells have reached the desired cell density in the matrix
or have confluently grown over most of the interstices of the cultured
matrix, the matrix is removed from the culture medium and either used in
the procedures to treat vascular smooth muscle cell hyperplasia described
below, or stored for subsequent implantation.
Alternatively, the matrix is seeded with the cells immediately prior
to implantation of the matrix.
Methods of Treating Intimal Hyperplasia
Patients can be diagnosed for vascular endothelial cell injury using
known methods, such as X-ray fluoroscopic examination of dye flowing
through a particular region of a blood vessel or other visual techniques,
the presence of symptoms such as pain, based on clinical judgment, or
signs evidenced physical examination. Alternatively, it can be assumed
that injury will arise due to performance of procedures such as
angioplasty, arterial bypass graft, peripheral bypass surgery, or organ
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transplantation and the patient treated based on the assumption that injt.~ry
or disease will inevitably arise.
In a preferred embodiment, endothelial cell-matrix strips are
applied to the site of injury during open field surgery. -In this
embodiment, the strips of cultured matrix are applied to the exterior of
the injured blood vessel over the internal site of injury, usually by
wrapping the matrix strips around the vessel. After implantation, the
cells remain viable and produce factors such as sulfated
glycosaminoglycan, including heparan sulfate.
If intimal hyperplasia had been observed prior to implanting or
wrapping the strips of matrices, the regression of hyperplasia is typically
evidenced by a decrease in pain or other symptoms of decreased blood
flow, or through the use of imaging techniques. The decrease in
hyperplasia or increase in flow rate through the injured vessel can be
monitored by the same methods used to initially diagnose the injury to the
vascular endothelium or blockage of the blood vessel.
The following non-limiting examples illustrate some of the various
aspects of compositions and methods used to treat vascular smooth muscle
cell hyperplasia and restenosis of blood vessels.
Example 1: Cell Engraftment and Characterization of Cells Cultured
in the Seeded Matrix.
Bovine aortic endothelial (BAE) and mutant Chinese hamster ovary
(CHO-745) cells were cultured on GelfoamT"' matrices. The mutant cells,
obtained from Dr. J. Esko (University of Alabama, Birmingham, AB),
served as control cells as they produce no demonstrable heparan sulfate
(Esko 1992') and, as a result, unlike endothelial cells, do not inhibit bFGF
binding to or mitogenesis of cultured vascular smooth muscle cells
(Nugent, et al., 19932). GelfoamTM has long been used as an implantable
surgical sponge and more recently as a scaffolding for cell growth
(Centra, et al., 19923). This material, isolated from porcine dermal
gelatin, was supplied in blocks (UpJohn Inc.), cut into 2.5 x 1 x 0.3 cm3
pieces and hydrated by autoclaving for 10 min in Hanks balanced salt
solution (HBSS). Upon cooling, the blocks were placed in 17 x 100 mm
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polypropylene tubes to which 2 mL of cell suspension (0.6 x 105
cells/mL) was added. The endothelial cells were suspended in Dulbecco's
modified Eagle's medium (DMEM) and the CHO-745 cells were
suspended in Ham's F-12 medium. Media was supplemented with 1 g/L
glucose and 10% calf serum.
Culture tubes were gently agitated to disperse the cells and then
incubated at a 45 angle, 37 C, humidified, 5% CO2 and 95 % air, for up
to 15 days. Growth medium was changed on days 3, 7 and 12. On days
0, 2, 4, 7, 10 and 15, the number of cells in the media (non-adherent
cells) and those attached to the GelfoamTM were determined using a
hemocytometer. Prior to counting the attached cells, the GelfoamT"'
blocks were washed four times with HBSS to remove non-attached cells
and serum. Collagenase (1 mg/mL) was used to digest the GelfoamT'r',
releasing all the cells from the matrix. An 80 L aliquot was removed
for cell counting and the viability was checked by trypan blue exclusion.
The results are shown in Figure 1. Each data point represents the
average cell number the standard error (SEM) of duplicate
determinations.
Cells lined the interstices of this three dimensional collagen-like
matrix (Centra, et al., 19923), and remained within the matrices without
migrating outward. Cell growth followed a pattern similar to that
observed on tissue culture polystyrene. Cell viability as evaluated by
trypan blue exclusion remained at 90 2.3% for the BAE cells and 93.1
1.7% for the CHO-745 over the course of the culture.
The preservation of the inimune identity of cells within GelfoamTM
blocks recovered 14 days after placement in tissue culture or around rat
carotid arteries was determined by immunostaining for the endothelial
marker von Willebrand's factor as described by Edelman, et al., J. Clin.
Invest. 89:465-471 (1992).
The amount of heparan sulfate in conditioned media produced by
cells cultured on GelfoamTM or tissue culture polystyrene was measured.
GelfoamTM films containing endothelial cells or CHO-745 cells were
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incubated in culture medium containing no antibiotics and no calf serum
for 24 hours at 37' C. As a control, identical GelfoamTM films without
cells were incubated in the respective media. The medium was collected,
centrifugedl (5,000 x g), dialyzed exhaustively against water, and
concentrated by lyophilization. Total sulfated glycosaminoglycan was
determined using dimethylmethylene blue (Farndale, et al., Biochimica et
Biophysica Acta 883:173-177 (1986), and the amount of heparan sulfate
was assessed after samples were treated with heparinase. The endothelial
and CHO-745 cells grown on GelfoamTM were also radiolabeled with
35SOq (100 Ci/mL, 24 h) to visualize the metabolic synthesis of heparan
sulfate by the cells. The medium was collected, centrifuged, and 3SS04-
proteoglycan separated from the free 35SO4 by vacuum filtration through
cationic nylon membranes (Rapraeger and Yeaman, Anal. Biochem.
179:361-365 (1989)). Filters containing 35S04-proteoglycan were
incubated iin nitrous acid (0.48 M sodium nitrite combined with 3.6 M
acetic acid) and counted to determine the amount of radioactivity
incorporated within heparan sulfate.
Engrafted cells remained viable with full retention of biochemical
secretory ability and biologic potency as well as immunoidentity. Cells
cultured on GelfoamTM produced nearly identical amounts of total sulfated
glycosaminoglycan and heparan sulfate as that produced when the same
cells were grown on polystyrene dishes. In addition, there was no
significant difference in the profile of the proteoglycan when resolved on
5% SDS-.PAGE. For both cells grown on GelfoamTM and tissue culture
polystyrene, the majority of the proteoglycan migrated as a band greater
than 600 kDa relative to protein standards. When compared to CHO-745
cells grown on GelfoamTM, the engrafted endothelial cells produced an
= 11.1-fold greater amount of glycosaminoglycan (4.1 0.3 microgram/106
cells/day for endothelial cells versus 0.37 0.02 microgram/106
cells/day for CHO-745 cells). While 29.1 % of the glycosaminoglycan
produced by the endothelial cells was heparan sulfate (1.2 0.05
micrograni/106 cells/day), CHO-745 cells produced no detectable heparan
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sulfate. Conditioned media from Gelfoam' engrafted endothelial cells
also inhibited both binding of luI-bFGF to heparan sulfate proteoglycan
and the mitogenic effect of bFGF on vascular smooth muscle cells in a
dose dependent fashion, as shown by Figure 2. In contrast, conditioned
media from CHO-745 cells had no effect on binding, and similarly no
effect on growth factor induced mitogenesis, as shown by Figure 2.
Example 2: Inhibition of Intimal Hyperplasia in Rats.
Implantation of Matrix Strips Containing BAE Cells into Rats.
The in vivo effects of engrafted cells in matrix strips were
evaluated in an endothelial denuding arterial injury model in Sprague-
Dawley rats. Strips of GelfoamTM gelatin matrix (2.5 x 1 x 0.3 cm3 each)
containing BAE cells or CHO-745 cells, or no cells, were wrapped
around carotid arteries denuded with a balloon catheter, and their effect
on smooth muscle cell proliferation and intimal hyperplasia was
determined two weeks later.
Endothelial denudation of the left common carotid artery in
Sprague-Dawley rats was performed with a 2 French Fogarty balloon
catheter introduced through an external carotid arteriotomy and passed
three times in its inflated state over the endothelium of the common
carotid artery (Edelman, et al., 19924, Clowes, et al., 19835, Edelman, et
al., 1990). Strips of Gelfoam' (2.5 x 1 x 0.3 cm3 each) containing BAE
cells, CHO-745 cells, or no cells were wrapped around denuded arteries.
The strip ends overlapped, ensuring complete encircling of the artery.
Fascial planes were sutured closed to further immobilize the devices.
To compare the results of anticipated physiologic secretion of
endothelial cell products to the pharmacologic dosing of a proven
endothelial-cell analog, hydrogel films were formulated (Nathan, et al.,
19947) to release heparin alone at a dose, 2.5 0.1 microgram/day. This
dose and mode of delivery has previously been demonstrated to maximize
heparin's inhibition of neointimal hyperplasia (Edelman, et al., 19906;
Edelman, et al., 1993g; Edelman and Karnovsky 19949).
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On the 14th post-operative day, animals were euthanized and
perfused clear via the left ventricle with Ringer's lactate solution followed
by immersion fixation with Camoy's fixative (60% methanol, 30%
chloroform, 10% glacial acetic acid). The location of the implanted
matrices was noted and the matrices recovered with the entire length of
the intact arteries. The carotid arteries were harvested and cut into five
equal segments, three including the GelfoamTM wrap and segments above
and below. Segments were paraffm embedded and 6 micron sections
obtained along the length of each segment.
After staining with hematoxylinleosin or verHoeff's elastin stain,
the intimal, medial and adventitial areas, the intima:media area ratio and
the percent of luminal occlusion were calculated using computerized
digital planumetry with a dedicated video microscope and customized
software. Cell proliferation was assayed using immunocytochemical
identification of proliferating cell nuclear antigenicity (PCNA) and the
thymidine analog 5-bromo-2'-deoxyuridine (BrdU, New England Nuclear,
Dupont Corp., Wilmington, DE) which had been injected
intraperitoneally, at 50 mg/kg body weight, 3 and 7 days post-surgery and
one hour prior to sacrifice.
Segments of the unwrapped balloon injured artery were compared
to Gelfoam" wrapped segments. Comparisons were made across
treatment groups and within treatment groups to allow each animal to
serve as its own control. Statistical comparisons were performed using
analysis of variance (ANOVA) and subsequent differences among groups
using Student's t test. Data was rejected as not significantly different if p
values of greater than 0.05 were observed. Data line fits were established
using a linear regression and correlation model.
The in vivo potency of the endothelial cell engrafts was retained in
addition to their in vitro effects. Balloon denudation of the carotid
arterial endothelium led to an increase in the ratio of the area of the
tunica intima to the area of the tunica media (I:M) to 1.44 0.16, as
shown by Figure 3. Arterial wall segments exposed to GelfoamTM
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containing endothelial cells showed minimal hyperplastic disease. Intimal
hyperplasia in arterial segments beneath endothelial cell grafted
GelfoamTM was reduced to 0.17 0.07. This modulating effect was
specific to endothelial cells. Control CHO-745 cell engrafts had no
statistically significant effect-on intimal hyperplasia (I:M 1.36 0.32)
compared to balloon injury, alone or empty GelfoamTM matrices (I:M
1.20 0.11), as shown by Figure 3.
Heparin has been identified as the gold standard inhibitor of
smooth muscle cell proliferation and intimal hyperplasia, in major part
because it resembles endothelial-cell-derived heparan sulfate proteoglycan.
Indeed the perivascular release of heparin alone from hydrogel films
reduced proliferation to an I:M of 0.55 0.11. This result, however,
was 3.2 fold less effective than the control exerted by engrafted
endothelial cells despite hydrogel release of heparin at twice the rate of
release of heparan sulfate proteoglycan from the endothelial implants.
GelfoamTM engrafted endothelial cells reduced proliferation as well
as intimal hyperplasia. The number of proliferating cells relative to the
total number of cells in the tunicae media and intima were used as an
index of proliferation. GelfoamTM implants with engrafted endothelial
cells caused a statistically significant decrease in cell proliferation in
both
the tunica intima (20.4 % reduction) and the tunica media (26.3 %
reduction) relative to empty GelfoamTM implants, as shown by Figure 4.
Heparin infusion also decreased proliferation but the effect was not
statistically significant at the number of animals and sections examined.
Relative to balloon injury alone, heparin infusion reduced BrdU
immunostaining in the tunica intima by 11.0 % and 12.2 % in the tunica
media, as shown by Figure 4.
There was no evidence that the implants induced cellular or
systemic rejection. The general health and appearance of the animals
with the implants was unchanged from controls. Rats subjected to balloon
injury alone gained 52.9 3.6 gms over the 14 day experimental period,
and those animals that received a cell-free implant hydrated in either
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DMEM or HAMS-F12 gained 48.0 7.3 gms. The animals implanted
with endothelial-seeded implants gained 51.3 2.8 gms and the animals
with CHO-745 laden implants gained 53.4 6.5 gms. Rats receiving
perivascular heparin gained 56.7 6.2 gms, indicating that the treatment
had no side effects.
The results demonstrate the engraftment of endothelial cells on
three dimensional biopolymer scaffoldings with preservation of cell
viability, normal growth characteristics, i.mmunologic markers,
biochemical activity and physiologic effects.
The biologic effects of the engrafted endothelial cells were cell-
specific and superior to the infusion of a single pharmacologic analog of
an endothelial product, heparin. Conditioned media from implant
engrafted endothelial cells, but not heparan sulfate proteoglycan-deficient
CHO-745 cells, inhibited bFGF binding to and mitogenesis for vascular
smooth miiscle cells in a dose dependent fashion. Si.milarly, only
endotheli,al cell engrafts inhibited intimal hyperplasia. The implant alone
or seeded with CHO-745 cells had no statistically significant effect on cell
proliferation or intimal hyperplasia. Heparin is one of the most effective
anti-proliferative agents for vascular smooth muscle cells, yet even when
released ai: twice the rate of heparan sulfate proteoglycan production by
the endot.helial cells, intimal hyperplasia and smooth muscle cell
proliferation were reduced 3.2 fold less effectively than with the
endothelial cell implants.
The control exerted by the endothelial cell implants appears to
result solely from the biochemical effects of the engrafted cells.
Immunostaining with endothelial cell-specific markers detected no
evidence for early recovery of endogenous endothelial cells or the
migration of engrafted cells from their biopolymer scaffoldings to the
arterial lining in any of the recovered arterial segments. The effects of
the implants were localized in a similar manner to the focal effects
observed with the perivascular release of other compounds. There was no
evidence of a systemic or local immune response or graft rejection.
CA 02222497 2006-07-12
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